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BI/CH 422/622ANABOLISM OUTLINE:Overview of PhotosynthesisKey experiments:

Light causes oxygen, which is from water splitting (Hill) NADPH made (Ochoa)Separate from carbohydrate biosynthesis (Rubin & Kamen)

Light Reactionsenergy in a photonpigmentsHOW Light absorbing complexes

Reaction centerPhotosystems (PS)

PSI – oxygen from water splittingPSII – NADPH

Proton Motive Force – ATP Overview of light reactions

Carbon Assimilation – Calvin CycleStage One – Rubisco

CarboxylaseOxygenaseGlycolate cycle

Stage Two – making sugarStage Three - remaking Ru 1,5P2

Overview and regulationCalvin cycle connections to biosyn.C4 versus C3 plantsKornberg cycle - glyoxylate

RubiscoCarboxylaseMechanism―

OverviewMg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolateintermediate

Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water

Photosynthesis

Recall another C-C cleavage of a b-keto acid?

C3-tetrahedral intermediate

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RubiscoCarboxylaseMechanism―

OverviewMg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolateintermediate

Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water

Photosynthesis

Recall another C-C cleavage of a b-keto acid?

C3-tetrahedral intermediate

• O2 competes with CO2 for the active site.~1 in every 3 or 4 turnovers, O2 binds

• The reactive nucleophile in the rubisco reaction is the electron-rich enediol form of ribulose 1,5-bisphosphate.• The nucleophile adds to O2 to form 3-

phosphoglycerate (same as in Calvin cycle) and 2-phosphoglycolate (2-PG).

–2-PG is metabolically difficult.–Salvaging its Carbons requires energy–As it is produced in appreciable amounts, an

elaborate pathway has been cobbled together.

PhotosynthesisFirst Stage of Calvin Cycle

Oxygenase Activity of Rubisco

This process is called PHOTO-RESPIRATION

3

decarboxylase

N5N10CH2

THFSerine OHMetransferase

The Glycolate Pathway* • Complex ATP-consuming process for the

recovery of C2 fragments from photorespiration. 1st Glycolate is made.• Uses three organelles • Loss of C as CO2 by mitochondrial

decarboxylation of glycine (see Pyr family)• Two 2-PGs are converted to Ser + CO2.• The Ser is cycled back to the chloroplast to

generate one 3-PGA.• Whole cycle costs an ATP per 1 3-PGA

converted back

PhotosynthesisFirst Stage of Calvin Cycle

*Don’t confuse with the glyOXYlate Cycle

2

2

2

2

2

111

1

1

1

1

1

Dealing with the oxygenase activity of rubisco

*

*called Glycine Cleavage Enzyme in bacteria

PhotosynthesisFirst Stage of Calvin Cycle

• 2-carboxyarabinitol 1-phosphate inhibits carbamoylated Rubisco.

– transition state analog of b-keto acid intermediate– synthesized in the dark in some plants

Rubisco is Inhibited by Several Criteria:1. pH

• In the stroma the pH is ~8 when light is on• pH decreases in the dark; rubisco inactive at low pH

2. CO2 (for the carbamoylation of Lys-210; also better at higher pH)3. NADPH (indirectly through rubisco activase)4. a “Noctural” Inhibitor

Regulation of rubisco activity

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Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation

– 3-phosphoglycerate kinase (3PGK)– glyceraldehyde 3-phosphate

dehydrogenase (GAPDH)

Rubisco

chloroplast isoforms

*Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo)

10 enzymes

Overall: 6 CO2 + 12 NADPH + 10 H2O + 18 ATP→ 2 glyceraldehyde 3-phosphate (GA3P) + 4 H+ + 12 NADP+ + 18 ADP + 16 Pi

(6)

(12)

(6)

(12)

(12)(12)

(12)

(12)

(12)

(2)

(10)

(6)

(6)

3-PGA Reduced to GA3P

PhotosynthesisSecond Stage of Calvin Cycle: making Glucose

DG°’ = +5.5 kcal/mol –1.8 kcal/mol +3.7 kcal/mol

ATP ADP

3-Phosphoglycerate 1,3-Bisphosphoglycerate

NADP+NADPH+H+

Glyceraldehyde3-phosphate

Twelve 3-phosphoglycerate + 12 ATP + 12 NADPH + 12 H+ à 12 glyceraldehyde 3-phosphate + 12 ADP + 12 NADP+ + 12 Pi

• Requires most of the ATP and NADPH from photosynthesis at ratio of 12:12• Reverse of the key reactions in glycolysis (except NADPH used rather than

NADH)– uses chloroplast isozymes of 3-phosphoglycerate kinase (3-PGK) and

glyceraldehyde 3-phosphate dehydrogenase (GAPDH)• Driven forward by high concentration of NADPH and ATP in the chloroplast

stroma; and the pulling of GA3P and Pi by the cycle and ATP synthase

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Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation

– 3-phosphoglycerate kinase (3PGK)– glyceraldehyde 3-phosphate

dehydrogenase (GAPDH)

Rubisco

chloroplast isoforms

*Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo)

10 enzymes

Overall: 6 CO2 + 12 NADPH + 10 H2O + 18 ATP→ 2 glyceraldehyde 3-phosphate (GA3P) + 4 H+ + 12 NADP+ + 18 ADP + 16 Pi

(6)

(12)

(6)

(12)

(12)(12)

(12)

(12)

(12)

(2)

(10)

(6)

(6)

Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation

– 3-phosphoglycerate kinase (3PGK)– glyceraldehyde 3-phosphate

dehydrogenase (GAPDH)

Rubisco

chloroplast isoforms

10 enzymes

(6)

(12)

(6)

(12)

(12)(12)

(12)

(12)

(2)

(10)

(6)

(6)

DHAP ⇌TPI

(12)Aldolase

• 12 glyceraldehyde-3-phosphate are produced during the Calvin cycle in the assimilation of 6 carbons into Glc.• 2/12 glyceraldehyde-3-phosphate products are pulled out of the cycle and used for Glc6P, then to other carbos.

Fru 1,6P2

⇌

Fru6PPi

⇌

Glc6P

Fructose-1,6-bisphosphatase

PGI

Reverse of glycolysis: GA3P converted to DHAP by triose phosphate isomerase (TPI); GA3P and DHAP converted to fructose 1,6-bisphosphate (Fru1,6-P2) via aldolase, the to Fru6-P via fructose 1,6-bisphosphatase, then Glc 6-P via phosphoglucoisomerase(PGI).

10/12 glyceraldehyde-3-phosphate products (30 carbons) MUST be used to regenerate 6 ribulose-1,5-bisphosphate (30 carbons).

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Stage 3: Regeneration of Ru 1,5-P2

PhotosynthesisThird Stage of Calvin Cycle

1. We have 3-carbon compounds and need 5-carbon compounds: put 2 together to make 6 (stop at Fru6P), then can pull 2 carbons from 6 (leaving 4) and with another 3 carbon compound make a 5-carbon sugar.

DO THIS TWICE. It will use 4 GA3P to make 2 Fru6P, then 2 more GA3P to make 2 5-carbon sugars (Xyl5P).

From 4 GA3P

transketolase

2 2 2 2

10àuse 6 GA3P4 GA3P à 2 Fru 6-P2 GA3P à 2 Xyl 5-P

Stage 3: Regeneration of Ru 1,5-P2

PhotosynthesisThird Stage of Calvin Cycle

1. We have 3-carbon compounds and need 5-carbon compounds: put 2 together to make 6 (stop at Fru6P), then can pull 2 carbons from 6 (leaving 4) and with another 3 carbon compound make a 5-carbon sugar.

DO THIS TWICE. It will use 4 GA3P to make 2 Fru6P, then 2 more GA3P to make 2 5-carbon sugars (Xyl5P).

From 4 GA3P

transketolase

2 2 2 2

10àuse 6 GA3P4 GA3P à 2 Fru 6-P2 GA3P à 2 Xyl 5-P

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Transketolase: exchange of 2C

from ketose to an aldose acceptor

•Contains thiazolium anion for nucleophilic attack on carbonyls of ketose

•Also used by:– pyruvate dehydrogenase in

acetyl CoA formation– pyruvate decarboxylase in

ethanol metabolism– a-ketoglutarate

dehydrogenase in CAC– transketolase in pentose

phosphate pathway

Photosynthesis

Uses Thiamine Pyrophosphate (TPP) as the Cofactor

e.g., fructose 6-phosphate

e.g., xyulose 5-phosphate

e.g., glyceraldehyde 3-phosphate

Aldose producte.g., erythrose4-phosphate

PhotosynthesisStage 3

2 22

4à use 2 GA3P2à0 GA3P left

2 2 2

2 from Fru6-P

2+2=4 6 6

6 Sedoheptulose 1,7-bisphosphate dephosphorylated to sedoheptulose 7-phosphate (S7P) via sedoheptulose1,7-bisphosphatase– This enzyme is unique to plants.

Finally, RuP phosphorylated to ribulose 1,5-bisphosphate by phosphoribulokinase– This enzyme is also unique to

plants and regulated by NADPH

sedoheptulose 1,7-bisphosphatase

transketolasealdolase

⇌ ⇌

2. We have two 4-carbon Ery 4-P and still have 4/12 GA3Ps. Do it again; aldol condensation with DHAP to get Sedoheptulose 1,7-bisphosphate (Sed 1,7-P2).3. If Sed 1,7-P2 loses the C7-phosphate, as a ketose, we can pull 2 carbons off and put on our remaining 2 ketoses (GA3P)

2

2 from Ery4-P

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PhotosynthesisStage 3

2 22

4à use 2 GA3P2à0 GA3P left

2 2 2

2 from Fru6-P

2+2= 4 6 6

6 Sedoheptulose 1,7-bisphosphate dephosphorylated to sedoheptulose 7-phosphate (S7P) via sedoheptulose1,7-bisphosphatase– This enzyme is unique to plants.

Finally, RuP phosphorylated to ribulose 1,5-bisphosphate by phosphoribulokinase– This enzyme is also unique to

plants and regulated by NADPH

sedoheptulose 1,7-bisphosphatase

transketolasealdolase

⇌ ⇌

2. We have two 4-carbon Ery 4-P and still have 4/12 GA3Ps. Do it again; aldol condensation with DHAP to get Sedoheptulose 1,7-bisphosphate (Sed 1,7-P2).3. If Sed 1,7-P2 loses the C7-phosphate, as a ketose, we can pull 2 carbons off and put on our remaining 2 ketoses (GA3P)

2

2 from Ery4-P

2

Stage 3: Addition of Phosphate from ATP to Ribulose-5-Phosphate Commits Product to Calvin Cycle

ribulose-5-phosphate epimerase

ribulose-5-phosphate epimerase

PhotosynthesisStage 3:

SummaryRegeneration Steps for

10/12 of Glyceraldehyde-3-phosphate Products of the

Calvin Cycle (Overview)

2 2

2

2

2

2

2 2

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PhotosynthesisStoichiometry and Energetics of CO2 Assimilation in the Calvin Cycle & NET Reaction for Photosynthesis:

6 CO2 + 12 NADPH + 12 H+ + 12 H2O + 18 ATP→1 C6H12O6 + 2 H+ + 12 NADP+

+ 18 ADP + 18 Pi + 18 H+

DG°’ = –38 kJ/mol

DG°’ = –10 kcal/mol

DG°’ = –56 kcal/mol6Ru5Pà12GA3P

2GA3Pà1Glc

10GA3Pà 6Ru5P

NET: –104/6 = –17.3 per CO2

This is 3:2 ATP:NADPH! (from 8 photons)For every NADPH, 4 photons needed∴ need 12x4=48 photons to make 1 Glc

Stage 1/2

Stage 2 (end)

Stage 3Stage 3

Stage 2 (end)

Stage 1/2

1 Pi

2 Pi

12 Pi

Glucose

Photosynthesis: Control by Light and CO2 to Glucose• The assembly of one molecule of glucose

requires the capture of roughly 48 photons.– H+ move from the stroma to the thylakoid– creates alkaline conditions in the stroma

• Accompanied by Mg2+ transport from thylakoid to stroma

• Enzyme for photosynthesis and CO2assimilation more active in the alkaline & high [Mg2+] conditions of the stroma

• This is a source of coordinated regulation by several enzymes: – Target enzymes regulated by NADPH, Mg2+, and

pH, or all three are:• Rubisco• fructose 1,6-bisphosphatase • seduloheptose 1,7-bisphosphatase• glyceraldehyde 3-phosphate dehydrogenase• ribulose 5-phosphate kinase

(phosphoribulokinase)

Photosynthesis

Glucose

How H+ and Mg2+

Movement Assist in Activation of

Photosynthesis and CO2

Assimilation

Rel

ativ

e A

ctiv

ity

Rub

isco

, Fru

1,6P

2ase

, Sed

1,7P

2ase

30

60

100

How are these regulated by NADPH?

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Target enzymes regulated by Fd:NADPH:fructose 1,6-bisphosphatase seduloheptose 1,7-bisphosphatase glyceraldehyde 3-phosphate dehydrogenaseribulose 5-phosphate kinase (phosphoribulokinase)Rubisco activase

If oxidized (Cys residues in Cys-Cys disulfide form) à enzymes are inactive.In light, photosystem I sends e– to ferredoxin, which sends them to thioredoxin, which donates

them to disulfide bonds to reduce them to free Cys.

PhotosynthesisPhotosynthesis: Control from Light and CO2 to Glucose

Excess reduced Fd is only available after its use for NADPH production

fructose 1,6-bisphosphatasesedoheptulose 1,7-bisphosphataseglyceraldehyde 3-phosphate dehydrogenaseribulose 5-phosphate kinase

Light Activation of FOUR Calvin Cycle Enzymes via Electron-Driven Reduction of Cys-Cys Crosslinks

Photosynthesis

• Plant cells: use 3-C intermediates for further synthesis – Glyceraldehyde 3-phosphate (G3P) is the most important one.– made from CO2, H2O, plus ATP and NADPH from photosynthesis

Assimilation of CO2 into Biomass